Randomized trial of benznidazole for chronic Chagas' cardiomyopathy

BACKGROUND:The role of trypanocidal therapy in patients with established Chagas' cardiomyopathy is unproven.METHODS:We conducted a prospective, multicenter, randomized study involving 2854 patients with Chagas' cardiomyopathy who received benznidazole or placebo for up to 80 days and were followed for a mean of 5.4 years. The primary outcome in the time-to-event analysis was the first event of any of the components of the composite outcome of death, resuscitated cardiac arrest, sustained ventricular tachycardia, insertion of a pacemaker or implantable cardioverter-defibrillator, cardiac transplantation, new heart failure, stroke, or other thromboembolic event.RESULTS:The primary outcome occurred in 394 patients (27.5%) in the benznidazole group and in 414 (29.1%) in the placebo group (hazard ratio, 0.93; 95% confidence interval [CI], 0.81 to 1.07; P=0.31). At baseline, a polymerase-chain-reaction (PCR) assay was performed on blood samples obtained from 1896 patients; 60.5% had positive results for Trypanosoma cruzi on PCR. The rates of conversion to negative PCR results (PCR conversion) were 66.2% in the benznidazole group and 33.5% in the placebo group at the end of treatment, 55.4% and 35.3%, respectively, at 2 years, and 46.7% and 33.1%, respectively, at 5 years or more (P<0.001 for all comparisons)...

Towards a paradigm shift in the treatment of chronic Chagas disease.

Treatment for Chagas disease with currently available medications is recommended universally only for acute cases (all ages) and for children up to 14 years old. The World Health Organization, however, also recommends specific antiparasite treatment for all chronic-phase Trypanosoma cruzi-infected individuals, even though in current medical practice this remains controversial, and most physicians only prescribe palliative treatment for adult Chagas patients with dilated cardiomyopathy. The present opinion, prepared by members of the NHEPACHA network (Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas/New Tools for the Diagnosis and Evaluation of Chagas Disease Patients), reviews the paradigm shift based on clinical and immunological evidence and argues in favor of antiparasitic treatment for all chronic patients. We review the tools needed to monitor therapeutic efficacy and the potential criteria for evaluation of treatment efficacy beyond parasitological cure. Etiological treatment should now be mandatory for all adult chronic Chagas disease patients.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Triatominae-Trypanosoma cruzi/T. rangeli: Vector-parasite interactions.

Of the currently known 140 species in the family Reduviidae, subfamily Triatominae, those which are most important as vectors of the aetiologic agent of Chagas disease, Trypanosoma cruzi, belong to the tribes Triatomini and Rhodniini. The latter not only transmit T. cruzi but also Trypanosoma rangeli, which is considered apathogenic for the mammalian host but can be pathogenic for the vectors. Using different molecular methods, two main lineages of T. cruzi have been classified, T. cruzi I and T. cruzi II. Within T. cruzi II, five subdivisions are recognized, T. cruzi IIa-IIe, according to the variability of the ribosomal subunits 24Salpha rRNA and 18S rRNA. In T. rangeli, differences in the organization of the kinetoplast DNA separate two forms denoted T. rangeli KP1+ and KP1-, although differences in the intergenic mini-exon gene and of the small subunit rRNA (SSU rRNA) suggest four subpopulations denoted T. rangeli A, B, C and D. The interactions of these subpopulations of the trypanosomes with different species and populations of Triatominae determine the epidemiology of the human-infecting trypanosomes in Latin America. Often, specific subpopulations of the trypanosomes are transmitted by specific vectors in a particular geographic area. Studies centered on trypanosome-triatomine interaction may allow identification of co-evolutionary processes, which, in turn, could consolidate hypotheses of the evolution and the distribution of T. cruzi/T. rangeli-vectors in America, and they may help to identify the mechanisms that either facilitate or impede the transmission of the parasites in different vector species. Such mechanisms seem to involve intestinal bacteria, especially the symbionts which are needed by the triatomines to complete nymphal development and to produce eggs. Development of the symbionts is regulated by the vector. T. cruzi and T. rangeli interfere with this system and induce the production of antibacterial substances. Whereas T. cruzi is only subpathogenic for the insect host, T. rangeli strongly affects species of the genus Rhodnius and this pathogenicity seems based on a reduction of the number of symbionts.

House-level risk factors for triatomine infestation in Colombia.

BACKGROUND: Chagas disease, transmitted domestically by triatomine bugs, is the most important vector-borne disease in Latin America. The association between triatomine infestation and housing characteristics was investigated based on a standardized survey in 41 971 houses in 15 Departments in Colombia. METHODS: Multivariate logistic regression was used to test for associations of two highly correlated infestation measures of infestation (householders reporting having seen triatomines inside the house, and sending triatomines to the survey team), with 15 household-level risk factors. Risks were measured relative to a reference category of houses with up to three inhabitants, area up to 50 m(2), unplastered adobe walls, thatch roof and no outbuildings or domestic animals. RESULTS: The probability of seeing triatomines was highest for households with over seven inhabitants (OR = 1.24, 95% CI 1.11-1.39), overhead storage space (OR = 1.16, 95% CI 1.03-1.32), grain shed (OR = 1.25, 95% CI 1.02-1.52), cats (OR = 1.27, 95% CI 1.14-1.42) and pigs (OR = 1.16, 95% CI 1.03-1.30). Lowest risks were in houses with wooden walls (OR = 0.46, 95% CI 0.34-0.61), fully plastered walls (OR = 0.78, 95% CI 0.68-0.88), roofs made of tiles (OR = 0.51, 95% CI 0.33-0.78) and flagstone floors (OR = 0.57, 95% CI 0.42-0.76). Results for householders returning triatomines support this set of risk factors, but with wider confidence intervals. CONCLUSIONS: Surveillance of a few easily assessed household characteristics provides an accurate, rapid assessment of house-level variation in risk. Measured effect sizes for specific structural characteristics could be used to maximize the cost-effectiveness of programmes to reduce vector infestation and interrupt Chagas disease transmission by improving house quality.

Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors.

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease.

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification.

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.

Chagas disease and human migration.

Human Chagas disease is a purely accidental occurrence. As humans came into contact with the natural foci of infection might then have become infected as a single addition to the already extensive host range of Trypanosoma cruzi that includes other primates. Thus began a process of adaptation and domiciliation to human habitations through which the vectors had direct access to abundant food as well as protection from climatic changes and predators. Our work deals with the extraction and specific amplification by polymerase chain reaction of T. cruzi DNA obtained from mummified human tissues and the positive diagnosis of Chagas disease in a series of 4, 000-year-old Pre-Hispanic human mummies from the northern coast of Chile. The area has been inhabited at least for 7,000 years, first by hunters, fishers and gatherers, and then gradually by more permanent settlements. The studied specimens belonged to the Chinchorro culture, a people inhabiting the area now occupied by the modern city of Arica. These were essentially fishers with a complex religious ideology, which accounts for the preservation of their dead in the way of mummified bodies, further enhanced by the extremely dry conditions of the desert. Chinchorro mummies are, perhaps, the oldest preserved bodies known to date.

Isolation of Trypanosoma cruzi DNA in 4,000-year-old mummified human tissue from northern Chile.

A segment of DNA unique to the kinetoplast of Trypanosoma cruzi was isolated from spontaneously mummified human remains from the coastal area of northern Chile at sites dated from 2000 BC to about AD 1400. Following rehydration of the desiccated human tissue samples of heart, esophagus, or colon, the samples were extracted and primers employed to bind to a 330 bp kinetoplast minicircle DNA sequence present in T. cruzi. This segment was then amplified using the polymerase chain reaction (PCR), and the target segment was visualized by gel electrophoresis. This method enables the identification of Chagas' disease in an ancient body in the absence of recognizable anatomic pathological changes.

Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA.

Several groups have recently developed molecular tests for the detection of Trypanosoma cruzi, the causative agent of Chagas' disease. Polymerase chain reaction (PCR) amplification of kinetoplast minicircle DNA sequences appears to be the most sensitive method. However, the specificity of PCR-based diagnostic methods was challenged when the complete sequence of Trypanosoma rangeli DNA minicircles was discovered. In the present study, we conducted. PCR experiments using the S35/S36 primers in Rhodnius prolixus and Balb/c mice with single and mixed infections of T. cruzi and/or T. rangeli. In single infections, the profile of each trypanosome was easily distinguishable in haemolymph, salivary gland and intestinal tissues and faeces of insect vectors. In mixed infections of anterior intestine (where T. rangeli is more predominant than T. cruzi), the DNA amplification profile of both parasites was observed simultaneously. Conversely, only the T. cruzi profile was observed in rectal ampulla (where T. cruzi is more abundant than T. rangeli). In mice with single infections of T. cruzi or T. rangeli, the profiles of amplified DNA were easily distinguishable in each case. The T. cruzi profile was dominant in most mixed infections, probably due to the fact that T. cruzi minicircles are more abundant and consequently compete more eagerly for annealing with the S35/S36 primers. In cases of mixed infections where T. rangeli was initially more abundant than T. cruzi, the specific T. rangeli 760 bp band was present for 7 days after infection and then this band and others ranging from 300 to 450 bp disappeared and only the typical T. cruzi 330 bp band remained. The S35/S36 primers used in polyacrylamide gel electrophoresis (PAGE) detected T. cruzi specifically, and prevented misdiagnosis due to the presence of T. rangeli. This technique can also be used to identify parasites in different stages of the infection (acute or chronic) in vertebrate hosts and to localize the parasites in the insect vector.

[New sources of lectins to differentiate between Trypanosoma cruzi y T. rangeli]. / Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli.

Extracts of 176 species of Colombian plant seeds, corresponding to 49 families and 147 genera, were tested for detecting agglutinins against human red blood cells from A+, B+ and O+ groups, dog, horse and rabbit. Extracts with haemagglutination activity were used for agglutination of Trypanosoma cruzi and T. rangeli. In addition the hemolymph of 16 native species of invertebrates were tested in the same conditions. Serial dilution of extracts were used for agglutination reactions. Both T. cruzi and T. rangeli epimastigotes showed agglutination with the extract of seven different species of plant seeds and with two types of haemolymph of invertebrates. The seeds of five plants exclusively agglutinated the epimastigotes of T. cruzi and thus can be used for the differentiation between culture forms of the trypanosomes. The secretion of the lung of a snail (Bulimus sp.) lysed entirely the epimastigotes of T. cruzi but did not affect T. rangeli forms. No extracts were found which agglutinated or lysed exclusively the epimastigotes of T. rangeli.

Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli / Further sources of lectins for differentiation between Trypanosoma cruzi y T. rangeli

Os extratos de 176 espécies de sementes de plantas colombianas, correspondentes a 49 famílias e 147 gêneros foram testados para detectar a presença de aglutininas frente a hemácias humanas dos grupos A+, B+, O+ e de cachorro, cavalo e coelho. Os extratos que apresentaram alguma atividade hemaglutinante, foram usados para testar a aglutinaçao de Trypanosoma cruzi e T. rangeli. Além disso, a hemolinfa de 16 espécies nativas de invertebrados foram testadas nas mesmas condiçoes. Diluiçoes seriadas dos extratos foram usadas para as aglutinaçoes. Ambas epimastigotas de T. cruzi e T. rangeli foram aglutinadas com os extratos de sete sementes de plantas diferentes e com dois tipos de hemolinfa de invertebrados. As sementes de cinco plantas aglutinaram exclusivamente as epimastigotas de T. cruzi podendo assim ser usadas para a diferenciaçao entre as formas de cultura desses tripanossomos. A secreçao do pulmao do caramujo (Bulimus sp.) lisou completamente as epimastigotas de T. cruzi mas nao afetou as formas de T. rangeli. Nao foram encontrados extratos que aglutinaram ou lisaram exclusivamente as epimastigotas de T. rangeli.

Phylogenetic analysis of mtDNA lineages in South American mummies.

Some studies of mtDNA propose that contemporary Amerindians have descended from four haplotype groups, each defined by specific sets of polymorphisms. One recent study also found evidence of other potential founder haplotypes. We wanted to determine whether the four haplotypes in modern populations were also present in ancient South American aboriginals. We subjected mtDNA from Colombian mummies (470 to 1849 AD) to PCR amplification and restriction endonuclease analysis. The mtDNA D-loop region was surveyed for sequence variation by restriction analysis and a segment of this region was sequenced for each mummy to characterize the haplotypes. Our mummies exhibited three of the four major characteristic haplotypes of Amerindian populations defined by four markers. With sequence data obtained in the ancient samples and published data on contemporary Amerindians it was possible to infer the origin of these six mummies.